Anti-transforming growth factor beta (TGF-beta) treated stem cell composition and method

ABSTRACT

The invention relates to stem cell compositions comprising anti-TGF-β treated stem cells which are viable for at least 14 days in culture without replication or differentiation and methods for rapid and long term in vitro hematopoiesis and in vivo hematopoietic reconstitution using such anti-TGF-β treated stem cells.

[0001] This application claims priority to U.S. Provisional applicationSerial Nos. 60/117,217 and 60/117,219, expressly incorporated byreference herein.

[0002] This work was supported in part by U.S. Government Agency andGovernment Grant No. R01 DK48708. Accordingly, the United StatesGovernment may have certain rights in this invention.

FIELD OF THE INVENTION

[0003] The present invention relates to stem cell compositions andmethods for preparing them in vitro or ex vivo by culturing stem cellsin medium containing anti-TGF-β antibodies in the absence of exogenouslyprovided cytokines. Such treatment facilitates the survival of long termrepopulating hematopoietic stem cells (LTR-HSC) within the culture forat least 14 days without replication or differentiation, the rapidengraftment of such LTR-HSC following in vivo administration to a mammalor the rapid proliferation of such LTR-HSC following transfer to invitro culture conditions effective to result in such expansion.

BACKGROUND OF THE INVENTION

[0004] The hematopoietic stem cell (HSC) is a pluripotent progenitorcell that has been characterized as a cell that is transplantable, canself-replicate or generate daughter cells that are destined to commit tomature cells of different specific lineages.

[0005] Self-replication of the most primitive HSC produces daughtercells that possess a long (possibly unlimited) clonal lifespan, whiledifferentiation of HSCs results in a loss of such multilineagepotential, and corresponding lineage commitment with a progressivereduction of their clonal lifespan. Previous studies indicated thatsurvival of HSC ex vivo in the absence of growth factors is limited,resulting in a complete loss of HSC after about 0.5-4 days in culture(Bartelmez S, unpublished data; Ploemacher RE et al. Stem Cells11:336-347, 1993; Li and Johnson, Blood 15;84(2):408-14, 1994).

[0006] Transplantation studies have shown that a single HSC canrepopulate the marrow of a lethally irradiated mouse, demonstrating thatself-renewal of HSC occurs in vivo, as indicated by transplantationstudies wherein a single HSC repopulated the marrow of animmunodeficient mouse (Smith, L G et al., Proc Natl Acad Sci USA 88,2788-92, 1991: Osawa M et al., Science 273, 242-245, 1996). In addition,repopulation of secondary (and tertiary) recipients, has beendemonstrated (Dick J E et al Cell 42, 71-9, 1985; Jordan C T et al.,Genes Dev 4, 220-32, 1990; Keller G and Snodgrass R J Exp Med 171,1407-18, 1990).

[0007] Transforming growth factor beta-1 (TGF-β1) is known to directlyand reversibly inhibit the initial cell divisions of long-termrepopulating hematopoietic stem cells (LTR-HSC) in vitro. (See, e.g.,Sitnicka E et al, Blood, 88(1):82-88, 1996 and Ploemacher R E et al.,Stem Cells 11(4):336-47, 1993.) The in vivo administration of TGF-62 tohumans to enhance the number of hematopoietic progenitor cells inperipheral blood has also been described. (See, e.g. U.S. Pat. No.5,674,843, issued Oct. 7, 1997.) The mode of action of the observedpleiotrophic effect of TGF-62 on stem and progenitor cells has beenattributed to TGF-62 as an inhibitor of cell proliferation or a mediatorof apoptosis.

[0008] Murine marrow cells treated with anti TGF-β antibody togetherwith IL-3, IL-6 and stem cell factor demonstrated a greater retroviraltransduction efficiency of progenitor (CFU-C) and long-term repopulatingcells than cells treated with IL-3, IL-6 and stem cell factor alone (YuJ et al., Gene Ther 5(9):1265-71, 1998).

[0009] Extensive studies have been described wherein HSC are cultured inthe presence in various combinations of cytokines as a means to increasethe number of HSC. In general, such culture conditions have causeddifferentiation of HSC and do not result in survival or increasednumbers of viable long term repopulating HSC (Li and Johnson, Blood15;84(2):408-14, 1994; Peters S O et al., Blood. 87(1):30-7. 1996;Yonemura Y et al., Proc Natl Acad Sci USA. 93(9):4040-4. 1996).

[0010] High-dose chemotherapy and/or radiation therapy together withbone marrow transplantation or transplantation of a cell populationenriched for hematopoietic stem cells are standard treatment regimensfor some malignancies, including, acute lymphocytic leukemia, chronicmyelogenous leukemia, neuroblastoma, lymphoma, breast cancer, coloncancer, lung cancer and myelodysplastic syndrome, as well as for othernon-malignant hematopoietic diseases, e.g. thrombocytopenia. Suchtreatments have shown promise in effective elimination of several typesof cancer, however in all cases the high doses also destroy bone marrowstem cells. In addition, bone marrow transplantation may play a majorrole in the emerging field of gene therapy.

[0011] Clinical trials are underway using such regimens for thetreatment of various cancers, including ovarian cancer, thymomas, germcell tumors, multiple myeloma, melanoma, testicular cancer, lung cancer,and brain tumors.

[0012] In addition, HSC have been demonstrated to be capable ofrepopulating non-hematopoietic tissues, including but not limited toliver (Petersen B E et al., Science 284:1168-70, 1999) and neuronaltissue (Bjornson C R R et al., Science 283:534-7, 1999).

[0013] Cell preparations enriched for hematopoietic stem cells generallycontain a low percentage of cells capable of long-term hematopoieticreconstitution. In general, culture conditions effective to promote thesurvival of hematopoietic stem cells include cytokines, which stimulatecell division and differentiation of the cells, diminishing their longterm repopulating capability. Frequently, as a result, in vivoadministration of such cell preparations does not result in rapidrepopulation of the host hematopoietic system. In particular, the slowrepopulation of the neutrophil and platelet compartments of thehematopoietic system may result in susceptibility to infection and/orcomplications due to poor blood clotting. In addition, once isolated,stem cell preparations are typically frozen in liquid nitrogen forsubsequent use and upon thawing the number of viable stem cells isfurther reduced.

[0014] Therefore, a need remains to develop techniques for maintainingstem cells in culture and for the use of such cells in both rapid andlong-term hematopoietic reconstitution.

SUMMARY OF THE INVENTION

[0015] The present invention addresses two significant problems in thefield of stem cell transplantation. As known in the art, hematopoieticstem cells capable of long term repopulation in vivo generally do notsurvive in culture without cell division, which usually results indifferentiation of the cells out of the stem cell compartment. Inaddition, the time required for in vivo repopulation of thehematopoietic system of a subject following in vivo administration ofsuch stem cells is sufficiently long that passive administration ofplatelets and neutrophils is often required to ensure the survival ofthe patient.

[0016] The present invention provides a composition of anti TGF-62antibody-treated stem cells capable of survival for at least 14 days invitro or ex vivo with continuous anti TGF-β antibody treatment, and amethod for obtaining the same.

[0017] The anti TGF-β antibody-treated stem cell compositions of theinvention provide a source of stem cells for rapid and sustainedrepopulation of the hematopoietic system of the subject. Thecompositions of the present invention provide a number of advantagesrelative to currently available stem cell preparations including: (1)hematopoietic repopulation which takes place at least 2-fold morerapidly following in vivo administration; (2) hematopoietic repopulationin vivo with a minimal number of cells, e.g., at least 10-fold fewerstem cells if antibody-treated; (3) sustained repopulation of thehematopoietic system of the subject for a clinically useful time; (4)stem cell proliferation in vitro which takes place at least 2-fold morerapidly following transfer to culture conditions effective to promotesuch proliferation; (5) stem cell proliferation in vitro with a minimalnumber of cells, e.g., at least 10-fold fewer stem cells ifantibody-treated; and (6) sustained stem cell proliferation in vitroresulting in generation of various lineages of hematopoietic cells forat least six months.

[0018] In one preferred aspect of the invention, the stem cells arehuman hematopoietic stem cells, characterized as lacking the expressionof lineage markers (lin-), and either (a) positive for cell surfaceexpression of CD 34 and KDR and negative for cell surface expression ofCD38 or (b) positive for cell surface expression of both CD 34 and Thy1.

[0019] In another aspect, the culture conditions effective to preservethe viability and differentiation state of said stem cells includeculture medium which contains from about 0.5 to 100 μg/ml of anti TGF-βantibody and lacks exogenously provided cytokines.

[0020] The invention further provides a method of obtaining a stem cellcomposition characterized by prolonged survival in culture whichincludes the steps of obtaining a population of cells enriched for stemcells and exposing the stem cells, ex vivo, to an anti TGF-β antibody,under culture conditions, and for a period of time, effective topreserve the viability and differentiation state of the cells.

[0021] A method for rapid in vivo repopulation of the hematopoieticsystem of a subject and a method for rapid proliferation of a stemculture in vitro are further provided by the invention. Such methodsinclude the steps of obtaining a population of cells enriched for stemcells and exposing the stem cells, ex vivo, to an anti TGF-β antibody,under culture conditions, and for a period of time, effective topreserve the viability and differentiation state of the cells, followedby either (a) readministering the anti TGF-β antibody treated stem cellsto the subject or (b) transferring the anti TGF-β antibody treated stemcells to culture conditions effective to result in the rapidproliferation of the cells, for in vivo and in vitro applications,respectively.

[0022] These and other objects and features of the invention will becomemore fully apparent when the following detailed description is read inconjunction with the accompanying figures and examples.

BRIEF DESCRIPTION OF THE FIGURES

[0023]FIG. 1 illustrates the survival of single sorted LTR-HSC in thepresence of anti-TGF-β1 antibody as indicated by the number of HSC perwell as counted on day 0, 1, 3, 4, 5, 6, 7, 10, 11, 14, 18 whenincubated in medium alone or medium containing 0.8-100 μg/ml of TGF-β1neutralizing antibody, ID.11.16;

[0024]FIG. 2 shows the relative repopulation lethally irradiated micewith LTR-HSC treated with either TGF-β1 neutralizing antibody, ID.11.16or IgG1K isotype control antibody, at various time points up to 9 monthspost-transplant;

[0025]FIG. 3 shows the % survival of lethally irradiated mice followingtransplantation of LTR-HSC treated with either anti-TGF-β1 neutralizingantibody (ID.11.16) or IgG1K isotype control antibody; and

[0026]FIG. 4 illustrates the relative survival of blast cells at day 7following culture of 0, 25, 50 or 100 lin-CD34+ baboon cells in mediumcontaining either anti-TGF-β1 neutralizing antibody (ID.11.16) or IgG1Kisotype control antibody in the absence of exogenously provided growthfactors.

DETAILED DESCRIPTION OF THE INVENTION

[0027] I. Definitions

[0028] The terms below, as used herein, have the following meanings,unless indicated otherwise:

[0029] As used herein, the term “a cell-population enriched forhematopoietic stem cells” refers to a cell population obtained using thepositive and negative selection techniques described herein, wherein thehematopoietic stem cells are LTR- or STR-HSCs.

[0030] As used herein, the terms “HSC expansion” and an “increasednumber of HSC” refer to an increase in the number of LTR- HSC andSTR-HSC.

[0031] As used herein, the terms “stem cell expansion” and an “increasednumber of stem cells” refer to an increase in the number of stem cellswhich are not necessarily HSC.

[0032] As used herein, “long term repopulating hematopoietic stem cells”or “LTR-HSC”, refers to hematopoietic stem cells that aretransplantable, and contribute to all lineages of hematopoietic cellsfor an undefined period of time, when transplanted into totallyimmunosuppressed recipients and do not undergo clonal extinction, asexemplified herein by murine LTR-HSC. The long term repopulating abilityof candidate hematopoietic stem cells may be evaluated in an in vivosheep model or an in vivo NOD-SCID mouse model for human HSC and normalimmunosupressed mice for murine HSC, respectively, as further describedherein.

[0033] LTR-HSC have been isolated and characterized in mice usingfluorescence-activated cell sorter (FACS) selection of densitygradient-enriched, lineage-depleted bone marrow cells which are negativefor expression of the CD34 antigen, positive for expression of the CD117(c-kit) antigen, and exhibit low-level binding of the DNA binding dye,Hoechst 33342 (Ho-33342) and the mitochondrial binding dye, Rhodamine123 (Rh-123), (Wolf, et al., 1993). The isolated cell population wasdemonstrated to be transplantable and capable of repopulating lethallyirradiated recipients, when transplanted together with unfractionatedbone marrow cells.

[0034] As used herein, the term “short term repopulating hematopoieticstem cells” or “STR-HSC”, refers to murine hematopoietic stem cells thatare transplantable and contribute to all lineages of hematopoietic cellsfor a period of from about one week to 6 months, then undergo clonalextinction. The STR-HSC population may be selected by FACS sorting andare phenotypically defined as light density gradient-enriched bonemarrow cells which lack the expression of lineage markers (lin-), arepositive for c-kit (CD 117), Sca1 and CD34, exhibit low-level binding ofthe DNA binding dye, Hoechst 33342 (Ho-33342) and high-level binding ofthe mitochondrial binding dye, Rhodamine 123 (Rh-123).

[0035] The term “clonal extinction”, as used herein refers to theterminal differentiation of a single hematopoietic stem cell and all theprogeny produced by clonal expansion of that cell, such that no moredaughter cells are produced from the initial clone.

[0036] The term “pluripotent hematopoietic stem cells” refers tohematopoietic stem cells, capable of differentiating into all thepossible cell lineages.

[0037] As used herein, the term “high proliferative potential colonyforming cells” or “HPP-CFCs”, as used herein relative to hematopoieticstem cells refers to murine or human cells that proliferate in responsevarious cytokines and other culture conditions. By way of example,murine HPP-CFC are produced by culture of murine HSC in the presence ofrat rSCF, mouse rIL-3 and human rIL-6. The cells proliferate insemi-solid media, such as agar or methyl cellulose or as single cells inliquid culture, and form macroclones which have a diameter greater than1 mm, generally having greater than 100,000 cells per clone with densemulticentric centers. This population includes all murine HSCs, however,not all HPP-CFC are HSCs, and the HPP-CFC assay is not a specific assayfor LTR-HSC. In contrast, low proliferative potential (LPP) clonescontain from 2 to 100,000 cells per clone.

[0038] As used herein, “lineage-committed hematopoietic stem cells” arehematopoietic stem cells that have differentiated sufficiently to becommitted to one or more particular cell lineages, but not all celllineages.

[0039] As used herein, the term “lin -” or “lineage-depleted”, refers toa cell population which lacks expression of cell surface antigensspecific to T-cells, B-cells, neutrophils, monocytes and erythroidcells, and does not express antigens recognized by the “YW 25.12.7”antibody. (See, e.g., Bertoncello I et al., Exp Hematol 19(2):95-100,1991.)

[0040] As used herein, the terms “develop”, “differentiate” and “mature”are used interchangeably and refer to the progression of a cell from astage of having the potential to differentiate into multiple cellularlineages to becoming a more specialized cell committed to one or moredefined lineages.

[0041] As used herein, the term “purified”, relative to hematopoieticstem cells refers to HSCs that have been enriched (isolated or purified)relative to some or all of the other types of cells with which they arenormally found in a particular tissue in nature, e.g., bone marrow orperipheral blood. In general, a “purified” population of HSCs has beensubjected to density gradient fractionation, lineage depletion andpositive selection for c-kit and Sca-1 expression in addition to lowlevel staining with both Hoechst 33342 and Rhodamine 123.

[0042] As used herein, a population of cells is considered to be“enriched” for human HSC if greater than 0.1% of the CD 34+ cells havean immunophenotype characteristic of human HSC, e.g., CD34+ CD38− KDR+;or CD34+ Thy1+.

[0043] As used herein, the term “hematopoietic cells”, refers to thetypes of cells found in the peripheral blood which are typically assayedas indicators of hematopoietic reconstitution and includes platelets,neutrophils, B lymphocytes and T lymphocytes.

[0044] As used herein, the terms “in vivo repopulation” and “in vivoreconstitution” refer to an absolute neutrophil count in the peripheralblood which is greater than 500/μl and an absolute platelet count whichis greater than 30,000/μl. It follows that “time to repopulation” and“time to reconstitution” refer to the amount of time following in vivoadministration of a cell preparation until the time that the absoluteneutrophil count in the peripheral blood is greater than 500/μl and theabsolute platelet count is greater than 30,000/μl.

[0045] As used herein, the terms “tumor” and “cancer” refer to a cellthat exhibits a loss of growth control and forms unusually large clonesof cells. Tumor or cancer cells generally have lost contact inhibitionand may be invasive and/or have the ability to metastasize.

[0046] As used herein “treatment” of an individual or a cell is any typeof intervention used in an attempt to alter the natural course of theindividual or cell. Treatment includes, but is not limited to,administration of e.g., a cellular or pharmaceutical composition, andmay be performed either prophylactically, or subsequent to theinitiation of a pathologic event or contact with an etiologic agent.

[0047] As used herein, the term “improved therapeutic outcome” relativeto a cancer patient refers to a slowing or diminution of the growth ofcancer cells or a solid tumor, or a reduction in the total number ofcancer cells or total tumor burden.

[0048] II. Hematopoietic Stem Cell Compositions

[0049] Cytokines

[0050] Recently, combinations of cytokines, including stem cell factor(SCF, or c-kit ligand), thrombopoietin (Tpo, c-mpl ligand), and theligand for the Flt3/Flk2 receptor (FL), have been shown to act directlyon HSC (Ogawa M et al., Stem Cells 15 Suppl 1, 7-11, 1997; Ku H et al.,Blood 87, 4544-51, 1996; Ramsfjell V et al., Blood 88, 4481-92, 1996;Sitnicka E et al., Blood 87, 4998-5005, 1996; Young J C et al., Blood88, 1619-31, 1996; Yoshida M et al., Br J Haematol 98, 254-64, 1997;Matsunaga T et al., Blood 92, 452-61, 1998). In addition, Tpo as asingle growth factor has been demonstrated to support survival andmodest proliferation of highly purified HSC in vitro (Ramsfjell V etal., Blood 88, 4481-92, 1996; Sitnicka E et al., Blood 87, 4998-5005,1997).

[0051] TGF-β, Anti TGF-β Antibodies and Stem Cells

[0052] TGF-β1 has been shown to directly and reversibly inhibit theinitial cell divisions of murine long-term repopulating hematopoieticstem cells (LTR-HSC) in vitro (Sitnicka E et al, Blood 1996 Jul.1;88(1):82-88). It follows that blocking the effects of TGF-β would beexpected to promote such initial cell divisions. However, the variousliterature references directed to the effect of TGF-β and anti-TGF-62antibodies on HSC do not provide consistent results. For example, theadministration of TGF-β to humans has been described as capable ofenhancing the number of hematopoietic progenitor cells in the peripheralblood. (See, e.g. U.S. Pat. No. 5,674,843, issued Oct. 7, 1997.) Inother references the effect of TGF-β on stem and progenitor cells hasbeen described as inhibition of cell proliferation or mediation ofapoptosis, based on the demonstration that if LTR-HSC were cultured withgreater than 0.1 ng/ml TGF-β1 (plus hematopoietic growth factors [HGF]),the probability of the maintenance or expansion of HPP daughter cellsappeared to increase (Sitnicka E et al, Blood 1996 Jul. 1;88(1):82-88).It was further observed that when a neutralizing anti-TGF-β1 monoclonalantibody was added with c-kit ligand or IL-3 to the cells, theproportion of LTR-HSC that divided increased as well as did the averageclone size (Sitnicka E et al, 1996).

[0053] Various references to culture of hematopoietic cells in thepresence of anti TGF-62 antibodies may be found in the literature. Therecited culture conditions vary considerably, however, in general thereferences describe bone marrow cells or stem cell-enriched cellpreparations cultured under conditions which include combinations ofcytokines.

[0054] In one example, TGF-β was added to ex vivo cultures of murinestem cells containing interleukin-3 (IL-3), IL-6, and stem cell factor(SCF) was shown to suppress short- and long-term repopulating activityin a murine competitive repopulation assay. An anti TGF-62 neutralizingantibody, reversed such effects relative to control cultures containingIL-3, IL-6, and SCF alone (Soma T et al., Blood 1996 Jun.1;87(11):4561-7).

[0055] Several other references describe murine marrow cells treatedwith anti TGF-β antibody together with various cytokines, e.g., IL-3,IL-6 and stem cell factor, wherein a greater retroviral transductionefficiency was observed in progenitor (CFU-C) and long-term repopulatingcells relative to cells treated with IL-3, IL-6 and stem cell factoralone. (See, e.g., Yu J et al., Gene Ther 1998 September;5(9): 1265-71).

[0056] In another example, Dexter-type long-term murine bone-marrowcultures were treated with a monoclonal antibody that neutralizes thebiological activity of TGF-β resulting in at least three times as manystem cells as control cultures (Waegell W O et al., Exp Hematol 1994October;22(11):1051-7).

[0057] Improved gene transfer into human hematopoietic progenitor cellsprestimulated with cytokines was demonstrated when the effect of TGF-β1was blocked by antisense or antiserum to release stem cells fromquiescence. (See, e.g., Hatzfeld et al., 1991, J. Exp. Med., 174, 925;Hatzfeld A et al., Hum Gene Ther 1996 Jan. 20;7(2):207-13; Imbert A M etal., Exp Hematol 1998 May;26(5):374-81; and U.S. Pat. No. 5,958,774.)

[0058] Such experiments are generally directed to releasing stem cellsfrom quiescence (i.e., causing them to enter the cell cycle and todifferentiate).

[0059] Numerous anti TGF-β antibodies are described in the literatureand many are commercially available. Table 1, below provides backgroundinformation on representative anti TGF-62 antibodies and a summary ofthe effect of each antibody on support for stem cell survival; theability of HSC treated with the antibody to induce rapid repopulationupon transfer in vivo or upon in vitro culture under conditions whichpromote HSC differentiation; the ability of HSC treated with theantibody to induce sustained repopulation upon such in vivo transfer orin vitro culture. TABLE 1 Monoclonal antibodies for treatment of HSC¹.Supportive Ab-treated HSC Ab-treated HSC Monoclonal of LTR-HSC inducerapid induce sustained Ab Antibody Immunogen survival repopulationrepopulation specificity ID11.16 Bovine TGF-β2 ++++ ++++ ++++ TGF-β1,TGF-β2 12H5 Human TGF-β1 ++ +++ ++++ TGF-β1 2G7 Human TGF-β1 No +++ +++TGF-β1, TGF-β2, TGF-β3 3C7.14 Human TGF-β2 No +/− +++ TGF-β2 20724 HumanTGF-β3 No +++ +/− TGF-β3 IgG1K N/A — +/− +/− None Isotype No N/A — +/−+/− N/A treatment

[0060] The results presented in Table 1 indicate the characteristics ofan anti TGF-β antibody for use in the compositions and methods describedherein. Such characteristics include, specific immunoreactivity withTGF-β, preferably TGF-β1 or TGF-β2, and the ability to modify stem cellsfollowing exposure of the cells for at least 20 minutes to mediumcontaining the anti TGF-62 antibody and lacking exogenously providedcytokines. Such anti TGF-62 antibody-treatment is effective to result in(1) stem cell survival in vitro at 37° C. or 4° C. for at least 14 days,(2) rapid hematopoietic repopulation following in vivo administration ofantibody-treated stem cells, (3) induction of sustained repopulationfollowing in vivo administration; (4) rapid stem cell proliferation invitro following transfer to culture conditions effective to promote suchproliferation; (5) stem cell proliferation in vitro with a minimalnumber of cells; and (6) sustained stem cell proliferation in vitroresulting in generation of various lineages of hematopoietic cells.

[0061] Such anti TGF-β antibody-treated stem cells therefore maintainthe ability to provide long term sustained hematopoietic reconstitution(in vitro and in vivo), and also exhibit the capability of short term invitro and in vivo repopulation, a quality which untreated stem cells donot possess.

[0062] It will be understood that any anti TGF-β antibody which exhibitsthe above-described characteristics finds utility in the methods andcompositions of the invention and that the invention is not limited tothe specific antibodies included in the examples described herein.

[0063] It will also be understood that any anti-stem cell antibody whichexhibits the above-described characteristics finds utility in themethods and compositions of the invention and that the invention is notlimited to the anti TGF-β antibodies described herein.

[0064] The conditions for prolonged stem cell survival in vitropresented herein as 37° C. or 4° C. for at least 14 days, are an exampleof typical culture conditions used by those of skill in the art toculture stem cells. The methods described herein are not limited to suchconditions and the present invention includes stem cell treatment withanti TGF-β antibodies at any temperature which results in anti TGF-βantibody-facilitated survival of the cells.

[0065] Such antibodies may include, but are not limited to, polyclonal,monoclonal, chimeric, humanized, single chain, Fab fragments andfragments produced by an Fab expression library. Antibodies, i.e., thosewhich block the biological effect of TGF-β on HSC, are especiallypreferred. See, e.g., Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, 1988, expressly incorporated herein by referenceherein.

[0066] Human or humanized antibodies are preferred for in vivoapplications and for treatment of cells to be readministered in vivo dueto the lack of potential for side effects which often result from animmune response to the antibody itself.

[0067] In one approach, transgenic animals (e.g. xenomice) may beproduced which are capable, upon immunization, of producing a fullrepertoire of human antibodies in the absence of endogenousimmunoglobulin production. In this approach, large fragments of both thehuman heavy and light chain Ig genes have been inserted into the mousegermline to create a mouse strain capable of producing a broadrepertoire of antigen-specific, fully human antibodies.

[0068] The xenomouse produces B cells expressing human heavy chain (hmu) and human K light chain (h K), or h mu and mouse lambda (m lambda)light chain. These mice produce significant quantities of fully humanantibodies with a diverse adult-like repertoire and, upon immunizationwith antigens, generate antigen-specific fully human monoclonalantibodies. (See, e.g., Jakobovits, A, et al., Ann N Y Acad Sci764:525-35, 1995; Jakobovits, A, Curr Opin Biotechnol 6(5):561-6,(1995).

[0069] Such xenogenic mouse-derived human monoclonal antibodies may nothave the correct Ig heavy chain for complement fixation in humans, e.g.,IgG1. In such cases, the antibody encoding mRNA from the xenogenic mousehybridoma may be used to obtain cDNA into which the appropriate cDNA forthe IgG1 heavy chain is inserted. This cassette may then be insertedinto an expression vector using procedures routinely employed by thoseof skill in the art, and subsequently for used in the production oftransgenic goats. Transgenic goats have been developed wherein induciblepromoters can trigger the expression of the protein encoded therein suchthat it is secreted into the milk of the goats. This procedure allowsfor relatively low cost production of large quantities of humanmonoclonal antibodies.

[0070] In one preferred embodiment, the anti-TGF-β monoclonal antibodiesof the invention comprise humanized antibodies or human antibodies.

[0071] Alternative methods of blocking the biological effects ofproteins are known in the art and fall within the scope of the presentinvention. For example, antisense oligonucleotides are frequently usedto control gene expression through complementary polynucleotides, i.e.antisense DNA or RNA, to the control, 5′ or regulatory regions of thegene encoding TGF-β. For example, the 5′ coding portion of thepolynucleotide sequence which codes for the protein of the presentinvention is used to design an antisense oligonucleotide of from about10 to 40 base pairs in length. Oligonucleotides based on thetranscription start site, e.g. between positions −10 and +10 from thestart site, are preferred and the antisense DNA oligonucleotide isdesigned to be complementary to a region of the TGF-β gene involved intranscription [Lee, et al., Nucl. Acids Res. 6:3073 (1979); Cooney, etal., Science 241:456 (1988); and Dervan, et al. Science 251:1360(1991)], thereby interfering with or preventing transcription and thesubsequent production of TGF-β. Accordingly antisense oligonucleotideseffective to block the expression of TGF-β, preferably unchargedantisense oligomers with modified (e.g., non-phosphodiester backbones)may be used in practicing the invention described herein.

[0072] Methods of Obtaining Hematopoietic Stem Cells

[0073] In adults, the large majority of pluripotent hematopoietic stemcells are found in the bone marrow. However, small but significantnumbers of such cells can be found in the peripheral circulation, liver,spleen and cord blood.

[0074] Human hematopoietic stem cells for use in the present inventionmay be derived from human bone marrow, human newborn cord blood, fetalliver or adult human peripheral blood, after appropriate mobilization.

[0075] The frequency of hematopoietic stem cells can be dramaticallyincreased by treatment of a subject with certain compounds includingcytokines. Such “mobilized” peripheral blood hematopoietic stem cellshave become an important alternative to bone marrow-derivedhematopoietic stem cells transplantation procedures primarily becauseengraftment is more rapid. (See, e.g., Tanaka, J, et al., Int J Hematol69(2):70-4, 1999.)

[0076] Such mobilization may be accomplished using for example, one ormore of granulocyte colony-stimulating factor (G-CSF), stem cell factor(SCF), thrombopoietin (Tpo), and a chemotherapeutic agent (i.e.,cyclophosphamide).

[0077] Numerous methods for human hematopoietic stem cellenrichment/isolation are known in the art and generally includeobtaining bone marrow, newborn cord blood, fetal liver or adult humanperipheral blood which contains hematopoietic stem cells. Once obtained,a hematopoietic stem cell population may be enriched by performingvarious separation techniques such as density gradient separation,immunoaffinity purification using positive and/or negative selection bypanning, FACS or magnetic bead separation. Following such enrichmentsteps, the cell population is further characterized phenotypically andfunctionally.

[0078] Previous studies have also demonstrated that primitivehematopoietic cells, characterized as high proliferative potentialcolony-forming cells (HPP-CFC, in vitro) may be isolated by selecting afraction of density gradient-enriched, lineage-depleted marrow cells,further selecting a cell population based on a single stepfluorescence-activated cell sorter (FACS) fractionation for cells thatbound low levels of the DNA binding dye, Hoechst 33342 (Hoechst^(lo))and low levels of the mitochondrial binding dye, Rhodamine 123(Rho^(lo); Wolf N S et al., Exp Hematol 21(5):614-22, 1993).

[0079] In one exemplary enrichment method, normal murine marrow cellsare processed using two pre-enrichment steps based on density gradientcentrifugation (e.g., using Nycodenz 1.080 g/ml, Nygaard, Oslo, Norway),followed by negative selection using Dyna1 beads coupled to myeloid andlymphoid specific monoclonal antibodies and positive selection by FACSsorting of cells based on staining with Rhodamine 123 (Rh), Hoescht3342(Ho) and antibodies to c-kit.

[0080] Once obtained, such candidate HSC may be characterized in avariety of in vitro and in vivo assays generally known in the art, asfurther described below. Such assays include, but are not limited to, anHPP-CFC assay, a single-cell HPP daughter cell assay, a single-cell IL-3response assay, a single-cell assay for time to the first cell division,a cobblestone area-forming cell assay and an in vivo limiting dilutiontransplant assay to quantitate STR- and LTR-HSC.

[0081] Recently, it has been shown that a defined subpopulation ofmurine HPP-CFC are transplantable and that a subpopulation of the cellsthat give rise to HPP-CFC are LTR-HSCs, which can replicate ex vivo, asshown by the results of in vitro LTBMC and in vivo repopulation studies.(See, e.g., Yagi M et al., Proc. Nat. Acad. Sci. 96:8126-8131, 1999).

[0082] Culture/Function of Hematopoietic Stem Cell Compositions

[0083] Hematopoietic stem cells have been historically defined astransplantable cells, capable of self-renewal which possess the abilityto generate daughter cells of any hematopoietic lineage.Lineage-committed progenitor cells are defined as more differentiatedcells derived from hematopoietic stem cells.

[0084] The phenotypic markers which characterize the hematopoietic stemcell have been the subject of extensive debate and numerouspublications. As yet, there is no consensus as to which markers aredefinitive for murine or human hematopoietic stem cells, however, themarkers for LTR-HSC and STR-HSC, as used herein, are provided above.

[0085] Functional readouts that have been used to detect andcharacterize hematopoietic stem cells include the ability to formcolonies under particular conditions in cell culture (in vitro), such asin the long term culture initiating cell (LTCIC) assay (Pettengell R etal., Blood 84(11):3653-9, 1994), long term bone marrow culture (LTBMC;Dexter T M et al., Prog Clin. Biol Res 148, 13-33, 1984) and the highproliferative potential-colony-forming cell (HPP-CFC) assay. (See, e.g.,Yagi M, et al., Proc. Nat. Acad. Sci. 96:8126-8131, 1999.) Furtherfunctional characterization includes in vivo assay for long-termrepopulating hematopoietic stem cells (LTR-HSC) and short-termrepopulating hematopoietic stem cells (STR-HSC), as further describedbelow.

[0086] LTBMC (Dexter T M et al., 1984) develop a complex adherentstromal layer containing a large variety of cell types, and can generatenonadherent (NA) hematopoietic cells for periods of several months.Hematopoietic stem cells are also often characterized functionally byactivity in the high proliferative potential colony-forming cell(HPP-CFC) assay, as defined above.

[0087] HPP-CFC are generally characterized by: (1) a relative resistanceto treatment in vivo with the cytotoxic drug 5-fluorouracil; (2) a highcorrelation with cells capable of repopulating the bone marrow oflethally irradiated mice; (3) their ability to generate cells of themacrophage, granulocyte, megakaryocyte and erythroid lineages, and (4)their multifactor responsiveness. (See, e.g., McNiece, I. K., Int J CellCloning 8(3):146-60, 1990).

[0088] Murine HSC

[0089] Preferred cytokines for the culture of murine hematopoietic stemcells include one or more of interleukin-3 (IL-3), interleukin-6 (IL-6),interleukin-11 (IL-11), interleukin-12 (IL-12) stem cell factor (SCF),fms-like tyrosine kinase-3 (flt-3), transforming growth factor-62(TGF-β), an early acting hematopoietic factor, described, for example inWO 91/05795, and thrombopoietin (Tpo).

[0090] Long-term,reconstitution of mice with murine LTR-HSCs followingcomplete immunosuppression has been shown to require the transplantationof unfractionated bone marrow cells together with less differentiatedlong term repopulating cells, in order to provide initial, albeitunsustained engraftment, such that the completely immunosuppressed hostmay survive until the long term repopulating cells differentiatesufficiently to repopulate the host. (See, e.g., Jones, R. J., et al.,Nature 347(6289):188-9, 1990). LTR-HSCs may take several months toeffectively repopulate the hematopoietic system of the host followingcomplete immunosuppression.

[0091] Methods have been developed to distinguish the cells of the donorand recipient in murine hematopoietic reconstitution studies, by usingdonor hematopoietic stem cells, congenic at the CD45 locus, defined asCD45.1 and recipient hematopoietic stem cells defined as CD45.2, suchthat monoclonal antibodies may be used to distinguish donor andrecipient cells, i.e., by FACS analysis and/or sorting. In suchdetection methods, the recipient is infused with sufficient CD45.2positive bone marrow cells to keep the mouse alive until differentiationof CD45.1 donor cells occur to an extent sufficient to repopulate thehematopoietic system of the recipient. Such methods may be used todifferentiate LTR-HSC from STR-HSC and donor cells from recipient cells.

[0092] Human HSC

[0093] Human HSC are initially characterized by immunophenotype, e.g.,as lineage negative and either (1) CD34+/Thy1+ or (2) CD 34+/CD38− cellsthat are also KDR+. Human HSC may also be characterized by telomerelength, where cells with high proliferative capacity have longertelomeres. In general, a population of cells is considered to beenriched for human HSC if greater than 0.1% of the CD 34+ cells have theimmunophenotype, CD 34+ CD38− KDR+ or CD34+ Thy1.

[0094] Preferred cytokines for the culture of human hematopoietic stemcells include one or more of interleukin-3 (IL-3), interleukin-6 (IL-6),interleukin-11 (IL-11), interleukin-12 (IL-12), stem cell factor (SCF),fms-like tyrosine kinase-3 (flt-3), transforming growth factor-62(TGF-β), an early acting hematopoietic factor, described, for example inWO 91/05795, and thrombopoietin (Tpo).

[0095] Human adult hematopoietic stem cells are mostly quiescent or slowcycling. However, it has been demonstrated that when human stem cellsare cultured under conditions which include exogenously providedcytokines, wherein TGF-β1 is blocked; quiescent, hematopoieticmultipotent progenitors grow in a short term culture assay in which thecells do not grow when TGF-β1 is not blocked.

[0096] III. Treatment of Murine HSC with Anti TGF-62 Antibodies.

[0097] Greater than 90% of single sorted murine LTR-HSC (lineage neg.,Ph^(low), Ho^(low), c-kit +, Sca-1 +) have been shown to form highproliferative potential (HPP) clones in the presence of SCF, IL-3, andIL-6 (Sitnicka E et al., Blood 87:4998-5005, 1996). In addition, suchstudies have indicated that essentially 100% of purified HSC cultured assingle cells undergo their first cell division if specific hematopoieticcytokine combinations are present, e.g., SCF (c-kit ligand) plus IL-6,IL-11, IL-12, or IL-3 (Sitnicka et al. 1996).

[0098] LTR-HSC have also been shown to express either an active cellsurface form and/or an active secreted form of TGF-β1 (Lucas C et al.,J. Immunol., 145:1415-1422, 1990). Such endogenously expressed TGF-β1 issufficient to arrest cell division if cultured in the presence of singlegrowth factors that have been identified as survival factors for singleLTR-HSC (Li and Johnson, Blood 15;84(2):408-14, 1994; Ploemacher R E etal. Stem Cells 11:336-347, 1993). In addition, greater than 90% ofLTR-HSC clones exhibited a high proliferative potential (HPP), which isdefined as clones able to attain greater than 100,000 cells by day 14 ofculture in response to SCF, IL-6 and IL-3; and are generallycharacterized by: (1) a relative resistance to treatment in vivo withthe cytotoxic drug 5-fluorouracil; (2) a high correlation with cellscapable of repopulating the bone marrow of lethally irradiated mice; (3)their ability to generate cells of the macrophage, granulocyte,megakaryocyte and erythroid lineages, and (4) their multifactorresponsiveness. (See, e.g., McNiece, I. K., Int J Cell Cloning8(3):146-60, 1990).

[0099] In general, LTR-HSC do not survive in culture without celldivision and/or differentiation and the survival of single LTR-HSCcultured in medium alone (without exogenously provided cytokines) islimited to a few days. The results presented herein demonstrate thatexposure of highly purified LTR-HSC to a neutralizing anti-TGF-betaantibody (e.g., ID.11.16, Celltrix Inc.) in the absence of exogenouslyprovided cytokines is effective to promote the survival of such LTR-HSCfor up to 18 days in culture without cell division or differentiation.(See Example 2.)

[0100] IV. Administration of Murine Anti TGF-β Antibody-Treated HSC toMice.

[0101] The results presented herein further demonstrate that suchexposure of highly purified LTR-HSC to a neutralizing anti-TGF-βantibody for a period of time from about 20-180 minutes dramaticallyreduces the time required for engraftment of LTR-HSC and that suchengraftment is sustained on a long term basis. The results of suchassays indicate that a substantial proportion of the surviving cellsretained their long term repopulating ability. (See Example 2.) Theseresults show that when stem cells are treated with anti-TGF-62 antibodyfor 20 or more minutes prior to in vivo administration, the treatedcells acquire the ability to rapidly repopulate the hematopoieticsystem, a quality typically attributed to short term repopulating cells.

[0102] In general, lethally irradiated mice kept under pathogen freeconditions will die by about day 12 following lethal irradiation (e.g.,at 950 rads), presumably due to a lack of platelets and/or neutrophils.The anti-TGF-beta antibody-treated stem cell compositions and methodsdescribed herein provide a means to rescue such lethally irradiated miceby in vivo administration of as few as about 60 anti-TGF-betaantibody-treated stem cells, which upon in vivo administration resultsin 100% survival of lethally irradiated mice at day 12 following lethalirradiation.

[0103] A demonstrable anti-TGF-beta antibody effect requires a cellpreparation enriched for LTR-HSC and cannot be seen by anti-TGF-betaantibody treatment of unfractionated bone marrow or any other cellpopulation which contains substantial numbers of short term repopulatingcells, along with the long-term repopulating cells.

[0104] V. In Vitro Treatment of Baboon HSC with Anti TGF-62 Antibodies.

[0105] Due to the genetic similarities between primates and humans,primates are an attractive model for the study of human hematopoiesis.In previous studies, when baboon bone marrow was treated in vitro withrecombinant human stem cell factor (SCF or c-kit ligand), SCF alone hadlittle effect on the growth of hematopoietic colony-forming cells butthe number of colonies formed in response to erythropoietin (Epo),interleukin-3 (IL-3), and granulocyte-macrophage colony-stimulatingfactor (GM-CSF) did increase suggesting an increase in hematopoiesis.This was confirmed when SCF, administered in vivo, resulted in anincrease in the number of erythrocytes, neutrophils, lymphocytes,monocytes, eosinophils, and basophils in the peripheral blood and anincrease in the cellularity and the number of colony-forming-unit-granulocyte-monocyte (CFU-GM) and burst-forming unit-erythroid(BFU-E) cells in bone marrow (Andrews RG et al., Blood 1991 Oct.15;78(8):1975-80).

[0106] Culture systems are under development for the expansion ofprimate HSCs that retain functional attributes of HSC and for genetransfer into CD34-enriched baboon marrow repopulating cells. (See,e.g., Medin J A et al., Ann N Y Acad Sci 1999 Apr. 30;872:233-40 andKiem H P et al., Blood 1998 Sep. 15;92(6):1878-86.)

[0107] As detailed in Example 3, when approximately 100, 50 or 25 baboonlin-CD34+ cells, characterized and sorted by FACS, were treated withmonoclonal anti-TGF-β antibody (ID11.16, Celltrix Inc.) in the absenceof exogenously provided cytokines, for a time period of 7 days, theproportion of surviving cells and the percentage of wells with viableblast cells was increased relative to lin-CD34+ cells treated with theisotype control antibody, IgG1K. (See also FIG. 4.)

[0108] In addition, when single cells of the above phenotype werecultured in the presence of anti-TGF-β antibody, the percentage ofviable cells at days 7 and 14 was also increased relative to celltreated with the isotype control antibody, IgG1K.

[0109] VI. In Vitro Treatment of Human HSC with Anti TGF-62 Antibodies.

[0110] The culture of human adult hematopoietic stem cells underconditions containing exogenously provided cytokines and an agenteffective to block TGF-β, has been observed to result in an increase inmultipotent progenitor cells in a short term culture assay relative tocells cultured in the absence of such an anti- TGF-β1 agent.

[0111] More recently, it has been shown that the expression of FLT3 andthe IL6 receptor (IL6-R) is decreased by TGF-β1 but rapidly up-regulatedby anti-TGF-β1 (Fortunel N et al., Cell Sci 1998 July; 111 (Pt13):1867-75). When purified human stem cells characterized as CD34+Thy1+were cultured in the presence of a neutralizing antibody against TGF-β1,the percentage of cycling cells, proliferation, and absolute number ofclonogenic progenitors increased in relative to cultures which were nottreated with anti-TGF-β1 antibody (Imbert AM et al., Exp Hematol 1998May;26(5):374-81).

[0112] As detailed in Example 4, when human HSC, characterized by FACSanalysis as CD34+ CD38^(low) were treated with monoclonal anti-TGF-βblocking antibody (1D11.16, Celltrix Inc.) in the presence of cytokines,for a time period as short as 6 hours, a greater number of CFU-Ccolonies, a greater number of HPP clones with more than 100,000 cells,increased c-kit expression and an increased number of cells that areactively cycling were detected, confirming that the addition ofanti-TGF-β antibody to cultures of human HSC releases multipotentprogenitors from quiescence with a significantly higher hematopoieticpotential than those activated by cytokines alone.

[0113] VII. Methods and Compositions of the Invention

[0114] Transplantation of hematopoietic stem cells derived fromperipheral blood and/or bone marrow is increasingly used in combinationwith chemotherapy and/or radiation therapy for the treatment of avariety of disorders including numerous forms of cancer. The percentageof cells in such cell preparations that are capable of rapid and/orlong-term hematopoietic reconstitution is very low.

[0115] In addition, due to the lack of a culture system for in vitro orex vivo preservation of stem cells, once obtained, stem cellpreparations are typically frozen in liquid nitrogen until used. Uponthawing, the viability and number of stem cells is further reduced.Therefore, a need exists to develop a means to preserve stem cells invitro or ex vivo following enrichment and to facilitate rapid expansionof the cells following in vivo administration to a subject or in vitrotransfer to culture conditions effective to promote expansion and/ordifferentiation.

[0116] Many cancer treatment regimens, result in immunosuppression ofthe patient, leaving the patient unable to defend against infection.Supportive care for immunosuppression may include protective isolationof the patient, such that the patient is not exposed to infectiousagents, administration of: antibiotics, antiviral agents and antifungalagents; and/or periodic blood transfusions to treat anemia,thrombocytopenia (low platelet count), or neutropenia (low neutrophilcount).

[0117] Current transplantation regimens that employ cell populationsenriched for hematopoietic stem cells and/or bone marrow transplantationalso suffer from an excessive lag time between transplantation andrepopulation of the-patient's hematopoietic system, in particularpatients often suffer from a deficiency in neutrophils and platelets.

[0118] Neutrophils are involved in defending the host against infection.Frequently, following a chemotherapy or radiation therapy, a patientwill suffer from insufficient neutrophil counts for time period of fromabout 3 to 4 weeks, or a longer time period resulting in increasedsusceptibility to infection.

[0119] Platelets are necessary for effective blood clotting at a site ofinjury. Frequently, following chemotherapy, radiation therapy,transplantation of a cell population enriched for hematopoietic stemcells or bone marrow transplantation, a patient will suffer from aninsufficient platelet count for a time period of from about 4 to 6weeks, or a longer time period resulting in the patient being easilybruised and excessive bleeding.

[0120] The invention is based on the discovery that culture of stemcells in the presence of anti TGF-β antibodies is effective to result inboth maintenance of cells having the phenotype and function of stemcells for an extended time in culture (e.g., at 37° C. or 4° C.),without cell division or differentiation, and the ability of such cellsto provide rapid and sustained repopulation of the hematopoietic systemof a host following in vivo administration or rapid expansion anddifferentiation of such stem cells following transfer to in vitroculture conditions effective to result in such expansion and/ordifferentiation.

[0121] While the mechanism is not part of the invention, it will beunderstood that such rapid stem cell expansion following treatment withanti TGF-β antibodies implies that the number of stem cells is alsoincreased by treatment with anti TGF-β antibodies.

[0122] Culture conditions for maintenance of stem cells by treatmentwith anti TGF-62 antibodies are described herein. However, it will beunderstood that the optimal survival of stem cells is dependent upon theamount and type of anti TGF-62 antibody added to the culture, the timeof exposure thereto and the purity and source of the stem cells (i.e.,bone marrow, mobilized peripheral blood or cord blood (murine versushuman), or human fetal liver.

[0123] In one aspect, the invention provides hematopoietic stem cellsthat are preserved in culture at 37° C. or 4° C., following treatmentwith anti TGF-β antibodies. Such a hematopoietic stem cell compositionfinds utility in a variety of applications, including, but not limitedto, preserving a population of hematopoietic stem cells ex vivo forsubsequent in vivo administration to a subject for purposes of (1) rapidand sustained hematopoietic stem cell replacement therapy, (2) reducingthe immune response to allogeneic transplants (ie, GVHD), (3) treatmentof autoimmune disease; (4) gene therapy and (5) treatment ofHIV-infection in a subject.

[0124] Once an anti TGF-β antibody-treated stem cell composition isprepared, the cells may be maintained in culture at 37° C. or 4° C. forat least 14 days without cell division or differentiation. In general,such an anti TGF-62 antibody-treated stem cell composition is maintainedin culture until use.

[0125] However, the calls may be frozen in liquid nitrogen and storedfor long periods of time, using standard conditions, such that they canlater be thawed and used, e.g., for administration to a patient. Thecells will usually be stored in a typical freezing medium, e.g., 10%DMSO, 50% fetal calf serum (FCS), and 40% cell culture medium.

[0126] Autologous hematopoietic stem cell transplantation has been usedto treat many solid tumors, including but not limited to, breast cancerand ovarian cancer. Prior to hematopoietic stem cell transplantation thepatient may or may not receive a chemotherapy regimen to reduce theamount of tumor present, generally followed by: (1) the collection ofthe patient's hematopoietic stem cells from either bone marrow ormobilized peripheral blood, (2) culture of hematopoietic stem cells inthe presence of cytokines or cryopreservation in liquid nitrogen, (3)high-dose chemotherapy administration intravenously (in most cases), and(4) reinfusion of the patient's hematopoietic stem cells (IV),approximately 48 hours after the chemotherapy administration iscomplete, and (5) further treatment of the patient with growth factorsto promote the differentiation of the hematopoietic stem cells andrepopulation of the patients hematopoietic system. In general, duringthis time the patient is immunocompromised and protective isolation isrequired.

[0127] Allogeneic hematopoietic stem cell transplantation has been usedto treat patients with leukemia, aplastic anemia, lymphomas (Hodgkin'sdisease and non-Hodgkin's lymphoma), and immune deficiency diseases. Anallogeneic hematopoietic stem cell transplantation protocol is similarto that used for autologous transplantation with the exception that inallogeneic transplantation, the donor and recipient must be matchedbased on the similarity of HLA cell surface antigens in order tominimize the immune response of both donor and recipient cells againstthe other.

[0128] Graft Versus Host Disease (GVHD)

[0129] GVHD is a frequent complication of allogeneic transplantation.About half of the patients undergoing an allogeneic bone marrowtransplant develop some GVHD, which is generally mild, but can be lifethreatening in some cases. In GVHD, the donor's cells attack therecipient's organs and tissue. Patients with GVHD have an increasedsusceptibility to infection and the skin, liver, and gastrointestinaltract may be attacked in GVHD.

[0130] GVHD is caused by T-cells, which recognize the patient's cells asbeing foreign. T-cells are able recognize differences based on humanleukocyte antigens (HLA). Even when the donor and recipient have similarHLA types, many minor markers differ between them except when the donorand recipient are identical twins. Hence, graft versus host disease(GVHD) is a potential problem and treatment to minimize the GVH responseis part of the therapeutic regimen for most transplants.

[0131] In the case of hematopoietic stem cell transplantation, suchtreatment often includes, T-cell depletion (i.e., by elutriation whichremoves T-cells based on density gradient centrifugation) alone, or incombination with hematopoietic stem cell enrichment by selection usingmonoclonal antibodies with hematopoietic stem cell markers, and drugtherapy for prevention of GVHD, e.g., by administration of cyclosporine(an immunosuppressive drug), alone or together with mehtotrexate.

[0132] In one aspect, the culture of hematopoietic stem cells underconditions described herein results in a hematopoietic stem cellcomposition that results in rapid and sustained repopulation of thehematopoietic system of the subject following readministration.

[0133] Stem cells within such an ex vivo expanded stem cell compositionlack immunological memory of self and non-self antigens, such thattransplantation of the hematopoietic stem cells into an allogeneic hostis unlikely to result in GVHD.

[0134] One exemplary therapeutic regimen involves ex vivo culture ofstem cells derived from a cancer patient in the presence of anti TGF-62antibodies, wherein stem cells are purified from an stemcells-containing cell population taken from the patient in a mannereffective to eliminate cancer-containing cells and the cells arecultured under the conditions described herein such that the number ofviable cancer-free stem cells are maintained in culture at 37° C. or 4°C. for a period of 14 or more days. This is followed by reinfusion ofthe anti TGF-β-treated stem cell composition into the patient resultingin rapid and sustained repopulation of the hematopoietic system of thepatient by about 1-3 weeks post-transplant. In many cases, thetherapeutic regimen also includes additional intervention such asradiation therapy and/or chemotherapy. The treatment may occur prior to,during or subsequent to re-infusion of ex vivo expanded stem cells.

[0135] Autoimmune Disease

[0136] As hematopoietic stem cells differentiate they are exposed to thevarious antigens present on the cells and tissue of the host andimmunological tolerance is established during T cell development withinthe thymus. In general, T cells that would be reactive to host proteinsdo not survive. However, in some cases, the immune system may recognizeself antigens as foreign resulting in an immune reaction against one ormore endogenous antigens, leading to an autoimmune condition or disease.

[0137] Exemplary autoimmune conditions include organ specific formswherein the immune response is directed against, e.g., the cells of theadrenal glands, causing Addison's disease, against the thyroid causingauto-immune thyroiditis (Hashimoto's disease) or against the beta cellsof the islets of Langerhans in the pancreas, resulting ininsulin-dependent diabetes mellitus; and non-specific forms wherein theimmune response is directed against an antigen that is ubiquitous, e.g.,an immune reaction against DNA, resulting in the disease systemic lupuserythematosus. Further examples include, Sjögren's syndrome, caused bythe production of auto-antibodies against salivary ducts, rheumatoidarthritis. Autoimmunity may be the result of attack by antibodies,T-cells or both.

[0138] The invention provides methods and compositions for the treatmentof autoimmune disease. In such methods, stem cells are obtained from apatient, followed by treatment of the patient with chemotherapy,radiation therapy or other means to deplete the patient of residualT-cells. The patients' stem cells or stem cells from an allogeneic donorare cultured ex vivo in the presence of anti TGF-62 antibodies resultingin maintenance of viable stem cells in vitro at 37° C. or 4° C. for aperiod of at least 14 days, followed by reinfusion of the antiTGF-β-treated stem cell composition into the patient resulting inrepopulation of the hematopoietic system of the patient by about 1-3weeks post transplantation. As the stem cells develop in the presence ofthe antigenic repertoire of the host, the newly developed T-cells shouldnot recognize host antigens as foreign and GVHD should not occur.

[0139] Such an in vitro anti TGF-β antibody-treated hematopoietic stemcell composition lacks immunological memory of self antigens, such thattransplantation of the stem cell composition finds utility intransplantation regimens for treatment of a patient with an autoimmunedisease, in order to minimize or eliminate the autoimmune condition.

[0140] It will be understood that such ex vivo hematopoietic stem celltreatment and re-infusion is generally used in combination withadditional therapeutic intervention to minimize the autoimmune responseof the patent's cells which are present prior to and duringhematopoietic stem cell isolation and in vitro stem cell anti TGF-βantibody treatment. Such additional treatment components includecompositions and procedures known in the art for the treatment ofautoimmune disease.

[0141] Gene Therapy Applications.

[0142] Gene therapy is a fast evolving area of medical and clinicalresearch. Gene therapy encompasses gene correction therapy, and transferof therapeutic, genes and is being applied for treatment of cancer,infectious diseases, multigenic diseases, and acquired diseases.

[0143] Exemplary disease targets include, but are not limited to cancersuch as prostate cancer, breast cancer, lung cancer, colorectal cancer,melanoma and leukemia; infectious diseases, such as HIV, monogenicdiseases such as CF, hemophilia, phenylketonuria, ADA, familialhypercholesterolemia, and multigenic diseases, such as restenosis,ischemia, and diabetes.

[0144] Given that hematopoietic stem cells have been demonstrated to becapable of maintaining their numbers in vivo without exhaustion, canrepopulate at least the entire hematopoietic system and that matureblood cells circulate throughout the body where a corrected gene productneeds to be delivered or a corrected gene product would cure aparticular deficiency (e.g., adenosine deaminase deficiency), stem cellsare an optimal vehicle for gene therapy.

[0145] The challenge of gene transfer into stem cells using retroviralvectors has been twofold: (1) cell division of stem cells is requiredfor proviral integration to occur (2) during stem cell divisionself-replication and not differentiation must be achieved. The presentdiscovery provides a means to achieve both requirements.

[0146] Cell transduction is possible in vivo, however, it is simpler andmore easily controlled ex vivo or in vitro, rendering ex vivo culturedhematopoietic stem cells extremely useful for therapeutic gene therapy.(See, e.g., Beutler E, Biol Blood Marrow Transplant 5(5):273-6, 1999;Dao M, Leukemia 13(10):1473-80, 1999.)

[0147] An exemplary therapeutic gene therapy regimen may include thesteps of obtaining a source of stem cells from a subject, stem cellenrichment or purification, in vitro or ex vivo stem cell expansion,transduction of stem cells with a vector containing a gene of interest,and reintroduction into a subject.

[0148] The anti-TGF-β antibody treated stem cells described hereinprovide a means to genetically modify stem cells under conditionslacking exogenously provided cytokines.

[0149] The transfer of genetic material into cells can be achieved byphysical and chemical methods or by the use of recombinant viruses. Inthe case of ex vivo transfer, chemical and physical methods such ascalcium phosphate, electroporation and pressure mediated transfer ofgenetic material into cells are often used. Several recombinant viralvectors which have been used for effective delivery of genes intomammalian cells include viral vectors, for example, retroviral vectors,adenovirus vectors, adenovirus-associated vectors (AAV), herpes virusvectors, pox virus vectors; non-viral vectors, for example naked DNAdelivered via liposomes, receptor-mediated delivery, calcium phosphatetransfection, electroporation, particle bombardment (gene gun), orpressure-mediated gene delivery. Various reports have been presentedregarding the efficacy of gene therapy for the treatment of monogenicdiseases, early stage tumors, and cardiovascular disease. (See, e.g.,Blaese R M, et al., Science 270, 475-480, 1995; Wingo P A, et al.,Cancer 82(6), 1197-1207, 1998; Dzau V, Keystone Symposium Molecular andCellular Biology of Gene Therapy, Keystone, Co. Jan. 19-25, 1998; andIsner J, Keystone Symposium Molecular and Cellular Biology of GeneTherapy, Keystone, Co. Jan. 19-25, 1998.)

[0150] Characterizing Stem Cell Compositions

[0151] The stem cell compositions described herein, may be evaluated,e.g., by conventional FACS assays for the phenotype of cells produced byin vitro culture or at various time points after in vivo administrationof stem cells.

[0152] Phenotypic analysis is generally carried out using monoclonalantibodies specific to the cell type being analyzed. The use ofmonoclonal antibodies in such phenotypic analyses is routinely employedby those of skill in the art for cellular analyses.

[0153] Hematopoietic stem cells are characterized phenotypically asdetailed above. Such phenotypic analyses are generally carried out inconjunction with biological (functional) assays for a given cell type ofinterest, for example; (1) hematopoietic stem cells, LTCIC, cobblestoneforming assays, and assays for HPP-CFC; (2) granulocytes or neutrophils,clonal agar or methylcellulose assays wherein the medium contains G-CSFor GM-CSF; (3) megakaryocytes, clonal agar or methyl cellulose assayswherein the medium contains Tpo, IL-3, IL-6 and IL-11; and (4) erythroidcells, clonal agar or methyl cellulose assays wherein the mediumcontains EPO and SCF or EPO, SCF and IL-3.

[0154] It will be understood that the exact nature of such phenotypicand biological assays will vary dependent upon various factors,including the source and degree of purity of the stem cell compositionunder evaluation.

[0155] In cases where a subject has been diagnosed as having aparticular type of cancer, autoimmune disease or other diseasecondition, the status of the condition is also monitored usingdiagnostic techniques appropriate to the condition under treatment.

[0156] VIII. Utility

[0157] The hematopoietic stem cell compositions described herein findutility in a variety of applications. For example, an in vitro or exvivo stem cell composition which has been treated with anti-TGF-betamonoclonal antibodies serves as a source of cells for rapid repopulationof a subject following in vivo administration and for rapid in vitroexpansion/differentiation following transfer to the appropriate cultureconditions. In addition, such anti-TGF-beta antibody treated stem cellsprovide a source of stem cells for various cellular transplantation andgene therapy applications.

[0158] Anti-TGF-beta antibody treated stem cells also find utility inrepopulating non-hematopoietic tissues in vivo, including, but notlimited to liver. Further uses include the use of anti-TGF-beta treatedstem cells to initiate in vitro cultures of expanded and/ordifferentiated stem cells for any of a number of uses for whichclinicians currently rely on cell preparation containing small numbersof stem cells which must be used soon after they are prepared.

[0159] Such an in vitro or ex vivo anti-TGF-beta antibody treated stemcell compositions also finds utility in both autologous and allogeneichematopoietic engraftment when readministered to a patient, where thecells are freed of neoplastic disease and graft-versus-host disease canbe avoided.

[0160] Alternatively, such an in vitro or ex vivo anti-TGF-beta antibodytreated stem cell composition may be used for gene therapy to treat anyof a number of diseases. In such cases, genetically modified stem cellscontaining a transgene of interest, e.g., directed toward a particulardisease target, are prepared in vitro and reinfused into a subject suchthat the cell type(s) targeted by the disease are rapidly repopulated bydifferentiation of cells in the stem cell composition followingreinfusion into the subject.

[0161] All patent and literature references cited in the presentspecification are hereby incorporated by reference in their entirety.

[0162] The following examples illustrate but are not intended in any wayto limit the invention.

[0163] Materials And Methods

[0164] Murine HSC Preparation And Culture with Antibodies

[0165] In general, murine LTR-HSC were purified from B6SJL mice(CD45.1+) by flushing cells from femurs of male B6.SJL-Ptprc^(a)Pep3^(b)/BoyJ (Ly5.1) (CD45.1) (B6.SJL) mice (Jackson Labs, Bar Harbor,Me.) with medium consisting of IMDM medium (Gibco BRL Life Technologies,Gaithersburg, Md.) supplemented with 20% heat-inactivated defined horseserum (HyClone Laboratories, Logan, Utah), 100 units/ml penicillin-10μg/ml streptomycin, 2 mM L-glutamine. Low density cells are enrichedusing 1.080g/ml Nycodenz separation medium (Nycomed Pharma AS OSLO,Norway), followed by isolation of the lin⁻cell population using DynalBead depletion employing lineage-specific monoclonal antibodies.Lin⁻cells are then incubated with Hoescht 33342 (Ho, final concentration10 mM) for 1 hr at 37 degrees and Rhodamine 123 (Rh, final concentration0.1 μg/ml) is added during the final 20 minutes (Wolf, et al., 1993).Cells were then labeled with phycoerythrin (PE)-conjugated anti-c-kitantibody (1 μg/ml final). Finally, propidium iodide (PI) is added (2μg/ml final concentration) for detection of dead cells and cells areanalyzed and sorted by FACS within 1-4 hours. Cell sorting has beenperformed on a FACStar Plus flow cytometer(or Coulter Elite or Ortho 50)equipped with dual argon lasers, and an automated cell delivery unit(ACDU). Cells are kept chilled at 4° C. with a recirculating water bath.Monochromatic light at 351-364 nm and 488 nm is used for Ho and Rh 123excitations, respectively. Forward light scatter is detected using488bp10 and ND 1.0 filters. Ho emission is detected using a 515 lpfilter in order to maximize signals from hematopoietic cells (Goodell etal; Bartelmez et al., unpublished observations). Rh 123 emission wasdetected using a 530 bp20 filter, PE emission using a 575 bp20 filter,PI emission using a 610 lp filter. Cells were gated using the followingsteps: first, forward light scatter and PI fluorescence are analyzed andviable cells (PI negative) were selected. Next, gates are set at thevarious percentages of Rh fluorescence using a 4-log amplifier: thelowest 10% (defined as Rh^(low)) and the middle 40% of the peak (definedas Rh^(high)). Then Rh^(low) and Rh^(high) cells analyzed for theirlinear Ho fluorescence and logrithmic PE-anti-c-kit receptorfluorescence. Rh^(low) and Rh^(high) were sorted as individual cellsinto 96-well plates or collected in bulk.

[0166] LTR-HSC enriched in this manner have been intensivelycharacterized and have the following phenotype: “lin-, Rh-123 low,Ho-33342 low, c-kit+, Sca-1+, Thy-1 low, CD-34 negative, AA 4.1negative” (Bartelmez S, et al., “Functional resolution of hematopoieticlong-term and short-term marrow repopulating stem cells in vitro,”submitted to Blood, 2000).

[0167] LTR-HSC, characterized as described above were sorted directlyinto phosphate buffered saline (PBS) for all experiments in whichLTR-HSC were transplanted at time 0 (T=0), or if the cells were to becultured, the LTR-HSC were sorted directly into basic culture medium forLTR-HSC which generally contains (1) Fisher's medium, 20% horse serum,10⁻⁶ M hydrocortisone (HC) or (2) IMDM medium plus 12.5% horse serum and12.5% fetal bovine serum, 10⁻⁶ M hydrocortisone or (3) serum-free medium(QBSF-58, Quality Biological Inc., Gaithersburg. Md.). In PBS or mediumcontaining cultures containing exogenously provided cytokines, theculture medium also contains one or more of interleukin-3,6,11,12(IL-3,6,11,12), stem cell factor (SCF), or thrombopoietin (Tpo). Inother words, in experiments in which cells were directly transplantedprior to short in vitro exposure to anti-TGF-β antibodies, suchtreatment took place in PBS, not in culture medium.

[0168] Antibodies, e.g., anti TGF-β antibodies are added to culturemedium at a concentration of from about 0.8-100 μg/ml.

[0169] Baboon HSC Preparation and Culture with Antibodies

[0170] In general, baboon HSC were purified from bone marrow aspirates.Bone marrow (BM) buffy coat cells were labeled with IgM monoclonalantibody 12-8 (CD34) at 4° C. for 30 minutes, washed, incubated with ratmonoclonal antimouse IgM microbeads (Miltenyi Biotec, Auburn, Calif.)for 30 minutes at 4° C., washed, and then separated using animmunomagnetic column technique (Miltenyi Biotec) according to themanufacturer's instructions. The purity of the CD34-enriched cells wasbetween 83% and 97%. The CD 34-enriched cells were then stained withmonoclonal antibodies 9.6 (CD2), 51.1 (CD 8), and 24.1 (CD10), G17.2 (CD4) G28.4 (CD40) 1F5 (CD20) and 5B12 (an antigen expressed by baboonneutrophils), the cells were washed and stained with anti-murineIgG-FITC conjugated antibodies to detect lineage positive cells and aPE-directly labeled anti-CD34 antibody directed against a differentepitope than that recognized by the 12.8 anti CD34 antibody. In thismanner, lin-CD34+ cells could be identified and sorted by FACS.

[0171] The HSC were characterized by FACS analysis as lin-CD34+ andcultured in IMDM medium containing 12.5% horse serum and 12.5% fetalbovine serum, hydrocortisone and P/S. In cultures containing exogenouslyprovided cytokines, the culture medium also contained one or more of thefollowing purified recombinant human growth factors: SCF, IL-3, 6,G-CSF, GM-CSF, Tpo, and/or erythropoietin (Epo)

[0172] Antibodies, e.g., anti TGF-62 antibodies are added to culturemedium at a concentration of from about 0.8-100 μg/ml.

[0173] Human HSC Preparation and Culture with Antibodies

[0174] Human HSC were purified from umbilical cord blood samplescollected immediately after delivery. However, it will understood thathuman HSC may be obtained from other sources such as mobilizedperipheral blood or bone marrow as further described above. In general,CD34+ cells were purified using of immunomagnetic beads (Dyne1),suspended in PBS/BSA (0.2%) and incubated with an anti-CD34 fluoresceinisothiocyanate (FITC)-conjugated monoclonal antibody (mAb) (8G 1 2clone; Becton Dickinson, San Jose, Calif.) and an anti-CD38phycoerythrin (PE)-conjugated mAb (HB-7 clone; Becton Dickinson) for 30minutes at 4° C., then washed twice. Isotype non-specific FITC- and PE-IgG1 were used as negative controls. The CD38^(low) subpopulation wasdefined as the 10% of CD34+ cells with the lowest intensity of CD38antigen expression. The CD34+ CD38^(low) cell population was isolated byFACS and deposited into 96- we11 plates containing medium using aVantage fluorescence activated cell sorter (FACS; Becton Dickinson)equipped with an automatic cell deposition unit.

[0175] HSC were characterized by FACS analysis as the CD34+ CD38^(low)cell population and cultured in semi-solid or liquid medium as furtherdescribed in Example 4.

[0176] In cultures containing exogenously provided cytokines, theculture medium also contained one or more hematopoietic growth factorssuch as SCF, Tpo, IL-6, TGF-beta, IL-11, IL-12, flt-3, and IL-3. Ingeneral, hematopoietic growth factors were purchased from Peprotech,with the exception of TGF-β (Gift of Bristol-Meyers Squibb). Cytokineswere used at concentration of 10 ng/ml for IL-6, IL-3, GM-CSF and TGF-βand 50 ng/ml for SCF and flt-3, respectively.

[0177] The monoclonal anti-TGF-62 blocking antibody (ID11.16, CelltrixInc.) and the non-blocking 2G1.2 antibody were used at 20 μg/ml.

[0178] Immunophenotyping of LTBMC Cells.

[0179] LTBMC cells were centrifuged and resuspended in 1% (w/v) bovineserum albumin in Dulbecco's phosphate-buffered saline.Fluorochrome-conjugated monoclonal antibodies to various mouse CDantigens, or biotinylated anti-mouse CD34 and FITC- or PE-conjugatedstrepavidin (Pharmingen, San Diego, Calif.) were incubated with thecells on ice (1 μg antibody/1-2×10⁵ cells). Cells were washed andanalyzed by flow cytometry (FACScan, Becton-Dickinson, Mountain View,Calif.) in the presence of propidium iodide to exclude dead cells.

[0180] Clonogenic Cell Assays.

[0181] Colony formation assays were performed in soft agar cultures(murine) or methylcellulose (human) in the presence of recombinantcytokines (R & D Systems or PeproTec, Rose Hill, N.J.) (Sitnicka E etal., Blood 87, 4998-5005, 1996). Two to five thousand cells were addedper ml of culture and plated in 35 mm dishes. Cultures were incubatedfor 12 days, and colonies were counted using an inverted microscope. Insome experiments, cells were plucked from colonies and their morphologyassessed after staining with Giemsa. Cytokines were used at thefollowing concentrations: for CFC, 5 ng/ml mouse GM-CSF and 10% (v/v)L929 supernatant (mouse M-CSF); for HPP-CFC, 50 ng/ml rat SCF, 20 ng/mlhuman IL-6, and 10 ng/ml mouse IL-3.

[0182] Colony formation assays for murine versus human CFC differ inthat human HPP-CFC are carried out in methylcellulose medium (Stem CellTech., Cat. No. H4435), in the presence of SCF (50 ng/ml), IL-3 (50ng/ml), IL-6 (20 ng/ml), erythropoietin (EPO, 1 unit/ml) and GM-CSF (5ng/ml). (See, e.g., Andrews R G et al., J Exp Med 172(1):355-8, 1990.)

[0183] LTBMC assay conditions for human cells generally includecommercially available media, e.g., Fishers medium; horse serum(Hyclone, Logan, Utah) from a lot selected based on optimal HSCgeneration in murine Tpo-LTMC assays; purified recombinant human Tpo(rhuTpo, Genentech, South San Francisco, Calif.); hydrocortisone; ahuman stromal cell component which includes, but is not limited to,cells of mesenchymal origin, including fibroblasts, adipocytes,endothelial cells; and megakaryocytes.

[0184] Transplants and Competitive Repopulation Assays.

[0185] Cells from B6.SJL mice (CD45. 1) were harvested, washed, and usedunfractionated for transplant. For each test sample, 2-10 recipientC57Bl6 mice (CD45.2) were irradiated (950 rad, ¹³⁷Cesium source) andtransplanted by injection via the tail vein with the indicated number oftest cells mixed with 4×10⁵ fresh unfractionated CD45.2 marrow cells.Animals were maintained in microisolator cages in an SPF facility.Peripheral blood samples were obtained by retroorbital bleeding atvarious times post-transplant. Expression of the donor CD45.1 allele andlineage specific antigens was assessed by two-color flow cytometryanalysis of peripheral blood leukocytes using directly labeledmonoclonal antibodies as described above for cultured cells. Thefrequency of long-term repopulating units was estimated using themaximum likelihood model that requires limiting dilution celltransplants of the test cells (Taswell C., J Immunol 126, 1614-9, 1981).

[0186] In Vivo Assays for Human HSC.

[0187] In vivo assays for human HSC may be carried out by usingapproximately 10,000-20,000 purified lineage negative CD34+ cellsderived from culture in an in utero fetal sheep assay (Zanjani E D etal., Stem Cells 13(2):101-11, 1995).

EXAMPLE 1 Maintenance of Murine LTR-HSC in Vitro Following Treatmentwith Anti-TGF-Beta Mab

[0188] Single Cell Studies in the Absence of Exogenously ProvidedCytokines: HSC Survival Detected by Viability and HPP (macroclone) Assay

[0189] Anti-TGF-β1 antibody treatment of single LTR-HSC cultured in theabsence of exogenously supplied cytokines resulted in survival of a highproportion of cells up to 18 days as single cells compared to LTR-HSCcultured in medium alone in which single cell survival was limited to afew days. FIG. 1 depicts the survival of single sorted LTR-HSC asindicated by the number of HSC per well counted on day 0, 1, 3, 4, 5, 6,7, 10, 11, 14, and 18 when incubated in medium alone or in mediumcontaining 0.8-100 μg/ml anti-TGF-β1 neutralizing antibody (ID.11.16,Celltrix Inc.), indicating that greater than 3-fold more HSC survived atleast 14 days in medium containing 20 μg/ml of the anti-TGF-β1neutralizing antibody relative to HSC incubated in medium alone. Theobserved survival effect was dependent on the concentration of theanti-TGF-β1 neutralizing antibody.

[0190] Following such treatment, essentially all single cells began celldivision synchronously and formed macroclones upon the addition of acombination of cytokines, SCF+IL-3+IL-6, which not only reflects themaintenance of their high proliferative potential (a characteristic ofmurine HSC) but also suggests that the cells became synchronized inrespect to cell cycle entry (in contrast to freshly isolated LTR-HSCthat are heterogeneous with respect to the time required to enter thecell cycle).

[0191] Multiple Cell Studies in the Absence of Exogenously ProvidedCytokines: HSC Survival Detected by Transplantation Assay

[0192] 100 LTR-HSC were cultured without growth factors but in thepresence of α-TGF-β1, for 5 days, then assayed in a competitiverepopulation assay, as detailed above. The results indicate that asubstantial proportion of the surviving cells retained their LTR ability(Table 2, below). In this experiment, LTR-HSC were directly FACS sortedinto 96-well plates at 100 cells/well containing medium plus eitherα-TGF-β1 neutralizing antibody (ID.11.16, Celltrix Inc.) or isotypecontrol antibody IgG1K at a final concentration of 20 μg/ml. After 5days, the cells were counted, assayed for HPP formation in agar andtransplanted together with 3×10⁵ support cells into lethally irradiatedrecipients. The recipient animals were analyzed for the presence ofdonor type cells in the peripheral blood by FACS analysis. The resultssuggest that anti TGF-β1 antibody treatment promotes the survival of theLTR-HSC. TABLE 2 The effect of α-TGF-β1 on in vitro survival and in vivorepopulation capability of Rh^(low) cells. Viable % Donor % Donor CellsRepopulation Repopulation per HPP/ (1.5 months (10.5 months ConditionsMouse Mouse post-trans.) post-trans.) 100 Rh^(low) cells 0.8 ± 0.8 0 0 0(5d culture in medium + isotype control Ab) 100 Rh^(low) cells 40 ± 6 33 ± 5 25 ± 20 22 ± 17 (5d culture in medium + α- TGF-β1, ID.11.16)

EXAMPLE 2 Rapid in Vivo Repopulation of Murine LTR-HSC Following inVitro Treatment with Anti-TGF-Beta Mab

[0193] In one experiment, LTR-HSC were purified from B6SJL mice(CD45.1+); and incubated ex vivo for 1 hour in either culture mediumalone or culture medium containing mouse α-TGF-β neutralizing antibodies(ID11.16, Celltrix Inc.), in the absence of exogenously suppliedcytokines.

[0194] The antibody-treated LTR-HSC were then transplanted into lethallyirradiated congenic strain C57B16 (CD45.2) mice along with 400,000unfractionated bone marrow competitor cells (CD45.2). Donor derivedneutrophils, peripheral blood B lymphocytes and peripheral blood Tlymphocytes were quantitated by FACS analysis.

[0195] The results of a representative experiment are shown below inTable 3, and indicate that LTR-HSC treated with α-TGF-β1 antibodies for1 hour in the absence of exogenously provided growth factors, rapidlyand substantially engraft lethally irradiated recipients. The resultsindicate that the greater degree of early donor engraftment by α-TGF-β1treated LTR-HSC is primarily due to the rapid production of donorneutrophils in the transplanted recipient. TABLE 3 ²In vivo repopulationof murine LTR-HSC following in vitro treatment with anti-TGF-beta Mab.Number of Time post- transplanted % donor % donor % donor B % donor TTreatment transplant cells cells neutrophils lymphocytes lymphocytes day0 - #1 4 weeks 100 ± 12  2 ± 1 (2) 0 (2) 82 ± 25 (2) 18 ± 25 (2) (none)day 0 #1 4 weeks 100 ± 12 42 ± 8 (4) 92 ± 5 (4)  7 ± 3 (4)  1 ± 2.1 (4)(medium + α- TGF-β1 Ab for 1 h)

[0196] In further experiments LTR-HSC were purified from B6SJL mice(CD45.1+); and treated ex vivo for varying lengths of time with one of anumber of types of antibodies including (a) mouse anti-TGF-betaneutralizing antibodies (b) non-neutralizing (but TGF-beta binding)antibodies, (c) isotype control antibodies, (d) antibodies known to bindto LTR-HSC, but not to TGF-beta (i.e., antibodies to c-kit, Sca-1, orCD45). The antibody-treated LTR-HSC were then transplanted into thecongenic strain C57B16 (CD45.2) along with 400,000 unfractionatedcompetitor cells (CD45.2) and donor derived peripheral blood T-cells,B-cells, monocytes and neutrophils were assayed by FACS analysis andtotal cell counts were performed.

[0197] By way of example, when LTR-HSC were treated with an IgG1Kisotype control antibody, the engraftment of 100 LTR-HSC occurredslowly, such that at 9 months post-transplant only 3.9% of the cells inthe peripheral blood were donor-derived CD45.1+ cells. (See FIG. 2.)

[0198] In contrast, when LTR-HSC were treated with α-TGF-β1 neutralizingantibody (ID.11.16, Celltrix Inc.) for 20-120 minutes the cells rapidlyengrafted to reach greater than 30% donor cells by 3 weeks withsustained engraftment for at least 9 months. Again, early engraftmentwas predominantly donor neutrophils followed by B-cells and then T-cellsby 1.5 months. The results show that the HSC transferred into the micenot only were able to rapidly repopulate the animals, but also werecapable of sustained repopulation characteristic of LTR-HSC. (See FIG.2.)

[0199] In the next example, experiments are described where LTR-HSC weretreated with neutralizing α-TGF-β 1 antibody (ID11.16, Celltrix Inc.)for 20 to 120 minutes and transplanted into lethally irradiated mice(950 rads) without support marrow in order to test the ability LTR-HSCto rescue lethally irradiated mice directly through a mechanism of rapidrepopulation in which donor platelets and neutrophils are producedusually within the first 1.5 weeks post transplant.

[0200] The percent post-transplantation survival was evaluated 30 dayslater, as shown in FIG. 3. When as many as 250 LTR-HSC were treated withIgG1K isotype control antibody prior to transplantation none of the micesurvived. In contrast, when 60 or 250 LTR-HSC were treated withneutralizing α-TGF-β1 antibody (ID11.16, Celltrix Inc.) prior totransplantation, approximately 65% or 100% of the mice survived,respectively.

[0201] Consistent with these results, transplantation of single LTR-HSCtreated with neutralizing α-TGF-β1 antibody (ID11.16, Celltrix Inc.)into lethally irradiated mice resulted in engraftment in greater than80% of the mice that received transplants. The rapid engraftment ofLTR-HSC did not impair the long term repopulating ability as measured bya sustained high percentage of donor chimeras after greater than 6months post-transplant.

EXAMPLE 3 Maintenance of Baboon LTR-HSC in Vitro Following Treatmentwith Anti-TGF-Beta Mab

[0202] The effect of α-TGB-62 antibody on baboon LTR-HSC cultured understandard conditions was evaluated using baboon lin-CD34+ cells preparedas described above. These compositions are enriched for both STR- andLTR- HSC. Groups of approximately 100, 50 or 25 lin-CD34+ cells weredirectly deposited following FACS sorting into 96 well plates containingIMDM medium supplemented 12.5%FBS and 12.5% HS, HC and P/S andanti-TGF-β1 antibody ID11 or IgG1K without exogenously provided growthfactors and incubated continuously in the presence of the antibodies.The results presented in Table 4 indicate that indicate that treatmentof CD34+ baboon cells with anti-TGF-β1 antibody in the absence of addedgrowth factors is effective to increase the proportion of survivingcells and the percentage of wells with viable blasts at day 7 relativeto CD34+ cells treated with the isotype control antibody, IgG1K. Theseresults indicate that anti-TGF-β1 antibody ID11 promotes the survival ofprimitive baboon hematopoietic cells similar to the effect observed whenmurine LTR-HSC are treated with anti-TGF-β1 antibodies. TABLE 4Lin-CD34+ baboon cells cultured in the absence of added growth factors:ID11.16, Celltrix Inc. vs. IgG1K isotype Proportion of Total numberTotal number surviving cells % positive wells Conditions of cells (day0) of cells (day 7) (day 7) (viable blasts) experiment #1 medium +isotype  90 ± 10 4 ± 3 4% (3/12) 25% control Ab IgG1K (20 μg/ml)medium + isotype 42 ± 4 4 ± 2 9% (4/12) 33% control Ab IgG1K (20 μg/ml)medium + isotype 18 ± 3 0.7 ± 1.2 4% (1/12) 8% control Ab IgG1K (20μg/ml) medium + α-TGF-β1 88 ± 8 34 ± 17 39% (12/12) 100% (ID11.16, 20μg/ml) medium + α-TGF-β1 38 ± 2 18 ± 12 47% (12/12) 100% (ID11.16, 20μg/ml) medium + α-TGF-β1 19 ± 2 4 ± 4 21% (6/12) 50% (ID11.16, 20 μg/ml)experiment #2 medium + isotype 85 ± 9 8 ± 7 9% (5/12) 42% control AbIgG1K (20 μg/ml) medium + isotype 38 ± 4 4 ± 3 11% (3/12) 25% control AbIgG1K (20 μg/ml) medium + isotype 19 ± 3 0 ± 0 3% (0/12) 0% control AbIgG1K (20 μg/ml) medium + α-TGF-β1 89 ± 9 34 ± 9  38% (10/10) 100%(ID11.16, 20 μg/ml) medium + α-TGF-β1 39 ± 2 23 ± 13 59% (10/10) 100%(ID11.16, 20 μg/ml) medium + α-TGF-β1 19 ± 2 4 ± 5 21% (4/10) 40%(ID11.16, 20 μg/ml)

[0203] The relative survival of blast cells at day 7 followinginitiation of cultures with 0, 25, 50 or 100 lin-CD34+ baboon cells inmedium containing either anti-TGF-β1 neutralizing antibody (ID.11.16) orIgG1K isotype control antibody in the absence of exogenously providedgrowth factors is presented graphically in FIG. 4. TABLE 5 Effect ofanti-TGF-β1 Neutralizing Antibody On The Survival Of IndividuallyCultured Baboon CD34+ Cells In vitro % of viable single cells and clonesConditions day 3 day 7 day 14 medium alone 57%  2%  0% medium + α-TGF-β178% 28% 10% (ID11.16)

[0204] The data presented in Table 5 show that the effect of theanti-TGF-β1 antibody ID11 or IgG1K is again specific to the anti-TGF-β1antibody ID11 and is also a direct effect on the enriched baboon cellcomposition. The survival of single cells was in the order of thatobserved in the multiple cell cultures.

EXAMPLE 4

[0205] Maintenance of Human Stem Cells In Vitro Following Treatment withAnti-TGF-Beta Mab

[0206] The effect of α-TGF-β antibody on human stem cells cultured understandard conditions in the was evaluated using human CD34+ CD38^(low)HSC, prepared as described above.

[0207] When stem cells were cultured in semi-solid methylcellulose media(Stem Cell Technologies), the media contained IL3, IL6, SCF, GM-CSF andFL plus or minus α-TGB-62 antibodies.

[0208] Liquid IMDM medium (BioWhitaker) with 20% horse serum was used toculture human stem cells. In general, 100 μl of medium containing IL3,IL6, SF, FL and GM-CSF plus or minus TGF-β or TGF-β antibodies was addedto culture wells of 96 well plates on day 0. 100 μl of twiceconcentrated medium containing cytokines was added after 7 and 14 days.In bulk cultures, stem cells were plated at 2×10⁵ cells/ml in 96-welltissue culture plates (Lux) in 200 μl of complete medium containingcytokines. Depending on the experimental condition, this control mediumwas supplemented with TGF-β1 or TGF-β1 blocking antibody, as indicatedin Table 6.

[0209] For clonal studies, the cells in each well were counted using aninverted Leitz Inverted microscope at day 10 and 21. Phenotypic analysisand staining was carried out using cells harvested from 96-well platecultures. Cells were labeled with FITC-anti-c-kit antibodies(Pharminogen), stained with propidium iodide and stained for cell cyclestatus, then analyzed using a Vantage flow cytometer. The pairedStudent's I-test was applied to determine the significance ofdifferences between mean values obtained under each treatment condition.TABLE 6 The Effect Of Anti-TGF-Beta On Primitive HematopoieticProgenitor Colony Formation. Conditions HPP CFU-C (media additives)Colony #³ Colony Size Colony # Colony Size experiment #1 cytokines 18100-200 59 50-100 cytokines +  39* 200-400 82 100-150  α-TGF-β1cytokines + isotype 15 100-150 63 50-100 control Ab experiment #2cytokines  7 100-150 38 50-100 cytokines +  28* 200-300 62 100-150 α-TGF- β1 cytokines + isotype  8 100-150 57 50-100 control Ab

[0210] TABLE 7 The effect of anti-TGF-beta on clonal frequency in singlecell assay Conditions Number cells per clone⁴ (media additives) <20,00020,000-100,000 >100,000 experiment #1 cytokines 11  32 7 cytokines +α-TGF-β  3* 23 25* (continuous)* cytokines + α-TGF-β1 (6 hours)  4* 2119* cytokines + isotype control Ab 10  24 4 experiment #2 cytokines 4 292 cytokines + α-TGF-β 0 17 11* (continuous)* cytokines + α-TGF-β1 (6hours) 1 14  9* cytokines + isotype control Ab 3 19 1

[0211] TABLE 8 Effect of anti-TGF-beta antibody on cell-surface c-kitreceptor modulation and cell cycle progression. Conditions (mediaadditives)⁵ c-kit expression Cell cycle experiment #1 (% cells > 1 log)⁶G1 S G2M cytokines 33.5 65 28 7 cytokines + α-TGF-β 48.3* 45 39 15cytokines + isotype control Ab 29 67 27 5 cytokines + TGF-β 20.7* 85 114

[0212] The results presented above show that treatment of humanCD34+CD38+ cells with anti-TGF-β antibodies for a time period as shortas 6 hours results in a greater number of HPP clones (more than 100,000cells) and a greater number of CFU-C colonies, plus increased c-kitexpression and an increased number of cells that are actively cycling.Thus, addition of anti-TGF-62 antibodies to cultures of human HSCstimulated with cytokines releases multipotent progenitors fromquiescence with a significantly higher hematopoietic potential thanthose activated by cytokines alone. In addition, this effect can beaccomplished with a 6 hour exposure suggesting that the effect isconfined to the initial CD34+CD38− cells in the culture and not thesubsequent daughter cells. Thus the effect appears to be HSC specificand occurs prior to cell division.

[0213] In summary, the results presented herein show or suggest that aconsistent effect is observed when stem cells isolated from a primarymurine, baboon or human source are treated with anti-TGF-betaantibodies.

It is claimed:
 1. A stem cell composition capable of rapid in vivorepopulation of the hematopoietic system of a subject comprising, anisolated stem cell treated with anti-transforming growth factor beta(TGF-β) antibodies under culture conditions effective to block theeffect of TGF-β on replication and/or differentiation of said stemcells.
 2. The composition of claim 1, wherein said stem cells are humanhematopoietic stem cells, characterized as lacking the expression oflineage markers (lin-), and either (a) positive for cell surfaceexpression of CD 34 and KDR and negative for cell surface expression ofCD38 or (b) positive for cell surface expression of both CD 34 and Thy1.3. The composition of claim 1, wherein said culture conditions effectiveto preserve the viability and differentiation state of said stem cellsinclude culture medium containing from about 0.5 to 100 μg/ml of antiTGF-β antibody.
 4. The composition of claim 1, wherein said cultureconditions effective to block the effect of TGF-β include culture mediumlacking exogenously provided cytokines.
 5. The composition of claim 1,wherein said rapid in vivo repopulation means following in vivoreadministration of said anti TGF-β antibody treated stem cells,hematopoietic reconstitution occurs at least 2-fold more quickly thanfor administration of the same number of stem cells which have not beentreated with said anti TGF-β antibody.
 6. A method of obtaining a stemcell composition characterized by prolonged survival in culturecomprising: (a) obtaining a population of cells containing stem cellsfrom a subject; (b) purifying said cell population in a manner effectiveto result in an enriched stem cell composition; and (c) exposing saidstem cells, ex vivo, to an anti TGF-β antibody, under cultureconditions, and for a period of time, effective to preserve theviability and differentiation state of said stem cells.
 7. The method ofclaim 6, wherein said enriched stem cell composition includes human HSC,characterized as lacking the expression of lineage markers (lin-), andeither (a) positive for cell surface expression of CD 34 and KDR andnegative for cell surface expression of CD38 or (b) positive for cellsurface expression of both CD 34 and Thy1.
 8. The method of claim 6,wherein said culture conditions effective to preserve the viability anddifferentiation state of said stem cells include culture mediumcontaining from about 0.5 to 100 μg/ml of anti TGF-β antibody.
 9. Themethod of claim 6, wherein said culture conditions effective to preservethe viability and differentiation state of said stem cells includeculture medium lacking endogenously supplied cytokines.
 10. The methodof claim 6, wherein said prolonged survival in culture means theviability and differentiation state of said stem cells is preserved fora period of time which is at least 2-fold longer than for stem cellswhich have not been treated with anti TGF-β antibody.
 11. The method ofclaim 6, wherein said prolonged survival in culture means the viabilityand differentiation state of said stem cells is preserved for at least14 days longer than that of stem cells which have not been treated withanti TGF-β antibody.
 12. A method for rapid in vivo repopulation of thehematopoietic system of a subject comprising: (a) obtaining a populationof cells containing stem cells from a subject; (b) purifying said cellpopulation in a manner effective to result in an enriched stem cellcomposition; (c) exposing said stem cells, ex vivo, to an anti TGF-βantibody, under culture conditions, and for a period of time, effectiveto preserve the viability and differentiation state of said stem cells;and (d) readministering said anti TGF-β antibody treated stem cells tothe subject.
 13. The method of claim 12, wherein said enriched stem cellcomposition includes human HSC, characterized as lacking the expressionof lineage markers (lin-), and either (a) positive for cell surfaceexpression of CD 34 and KDR and negative for cell surface expression ofCD38 or (b) positive for cell surface expression of both CD 34 and Thy1.14. The method of claim 12, wherein said culture conditions effective topreserve the viability and differentiation state of said stem cellsinclude culture medium containing from about 0.5 to 100 μg/ml of antiTGF-β antibody.
 15. The method of claim 12, wherein said cultureconditions effective to preserve the viability and differentiation stateof said stem cells include culture medium lacking exogenously providedcytokines.
 16. The method of claim 12, wherein said prolonged survivalin culture means the viability and differentiation state of said stemcells is preserved for a period of time which is at least 2-fold longerthan for stem cells which have not been treated with anti TGF-βantibody.
 17. The method of claim 12, wherein said prolonged survival inculture means the viability and differentiation state of said stem cellsis preserved for at least 14 days longer than for stem cells which havenot been treated with anti TGF-β antibody.
 18. The method of claim 12,wherein said readministering includes autologous stem celltransplantation.
 19. The method of claim 12, wherein saidreadministering includes allogeneic stem cell transplantation.
 20. Themethod of claim 12, wherein said readministering includestransplantation of genetically modified stem cells.
 21. A method ofpreparing stem cells for rapid in vitro proliferation, comprising: (a)obtaining a population of cells containing stem cells from a subject;(b) purifying said cell population in a manner effective to result in anenriched stem cell composition; (c) exposing said stem cells, ex vivo,to an anti TGF-β antibody, under culture conditions, and for a period oftime, effective to preserve the viability and differentiation state ofsaid stem cells; and (d) transferring said anti TGF-β antibody treatedstem cells to culture conditions effective to result in the rapidproliferation of such stem cells.
 22. The method of claim 21, whereinsaid enriched stem cell composition includes human HSC, characterized aslacking the expression of lineage markers (lin-), and either (a)positive for cell surface expression of CD 34 and KDR and negative forcell surface expression of CD38 or (b) positive for cell surfaceexpression of both CD 34 and Thy1.
 23. The method of claim 21, whereinsaid culture conditions effective to preserve the viability anddifferentiation state of said stem cells include culture mediumcontaining from about 0.5 to 100 μg/ml of anti TGF-β antibody.
 24. Themethod of claim 21, wherein said culture conditions effective topreserve the viability and differentiation state of said stem cellsinclude culture medium lacking exogenously provided cytokines.
 25. Themethod of claim 21, wherein said prolonged survival in culture means theviability and differentiation state of said stem cells is preserved fora period of time which is at least 2-fold longer than for stem cellswhich have not been treated with anti TGF-β antibody.
 26. The method ofclaim 21, wherein said prolonged survival in culture means the viabilityand differentiation state of said stem cells is preserved for a periodof time which is at least 14 days longer than for stem cells which havenot been treated with anti TGF-β antibody.